Detection of Circulating SARS-CoV-2 Variants of Concern (VOCs) Using a Multiallelic Spectral Genotyping Assay

Abstract

Throughout the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved, resulting in new variants, some of which possess increased infectivity, immune evasion, and virulence. Such variants have been denoted by the World Health Organization as variants of concern (VOC) because they have resulted in an increased number of cases, posing a strong risk to public health. Thus far, five VOCs have been designated, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529), including their sublineages. Next-generation sequencing (NGS) can produce a significant amount of information facilitating the study of variants; however, NGS is time-consuming and costly and not efficient during outbreaks, when rapid identification of VOCs is urgently needed. In such periods, there is a need for fast and accurate methods, such as real-time reverse transcription PCR in combination with probes, which can be used for monitoring and screening of the population for these variants. Thus, we developed a molecular beacon-based real-time RT-PCR assay according to the principles of spectral genotyping. This assay employs five molecular beacons that target ORF1a:ΔS3675/G3676/F3677, S:ΔH69/V70, S:ΔE156/F157, S:ΔΝ211, S:ins214EPE, and S:ΔL242/A243/L244, deletions and an insertion found in SARS-CoV-2 VOCs. This assay targets deletions/insertions because they inherently provide higher discrimination capacity. Here, the design process of the molecular beacon-based real-time RT-PCR assay for detection and discrimination of SARS-CoV-2 is presented, and experimental testing using SARS-CoV-2 VOC samples from reference strains (cultured virus) and clinical patient samples (nasopharyngeal samples), which have been previously classified using NGS, were evaluated. Based on the results, it was shown that all molecular beacons can be used under the same real-time RT-PCR conditions, consequently improving the time and cost efficiency of the assay. Furthermore, this assay was able to confirm the genotype of each of the tested samples from various VOCs, thereby constituting an accurate and reliable method for VOC detection and discrimination. Overall, this assay is a valuable tool that can be used for screening and monitoring the population for VOCs or other emerging variants, contributing to limiting their spread and protecting public health.

Design and Development of Molecular Beacon-Based Real-Time PCR Assays to Identify Clostridioides difficile Types of Main Evolutionary Clades

“…Although WGS represents the ideal approach to improve pathogen surveillance and provides precise data for a fast identification of outbreaks, this methodology still presents problems for its applicability due to elevated costs, execution time, and personnel expertise required [3,44,61,64]. Real-time PCR is an established, semi-automated, fast method used to detect and monitor pathogenic bacteria and viruses for both diagnostics and surveillance purposes [37,65].…”

Molecular and Clinical Epidemiology of SARS-CoV-2 Infection among Vaccinated and Unvaccinated Individuals in a Large Healthcare Organization from New Jersey

“…Even with availability of in-house sequencing, turnaround times for WGS may entail several days, or even longer for large numbers of samples. By contrast, PCR-based screening assays such as ours [ 9 ] can deliver informative results within a single day, without the need for library preparation, costly materials, or expensive instrumentation [ 64 , 65 , 66 ]. Moreover, screening data can also inform clinical decisions regarding the use of monoclonal antibodies, many of which have been rendered ineffective by successive accumulation of particular spike gene mutations [ 63 , 67 , 68 , 69 , 70 ].…”

“…Moreover, screening data can also inform clinical decisions regarding the use of monoclonal antibodies, many of which have been rendered ineffective by successive accumulation of particular spike gene mutations [ 63 , 67 , 68 , 69 , 70 ]. Various real-time PCR-based screening assays capable of detecting multiple VOCs have been described [ 64 , 66 , 71 , 72 , 73 ], but to our knowledge have not been utilized as extensively, except perhaps for the one described by Moisan et al, which was used to analyze 2221 patient samples [ 74 ].…”

An ARMS-Multiplex PCR Targeting SARS-CoV-2 Omicron Sub-Variants

“…In a recent study by Chrysostomou et al, the identification of Omicron sub-variants was achieved through the detection of a combination of deletions/insertion in the ORF1a and S gene regions using a uniplex molecular beacon-based real-time RT-PCR assay. This assay correctly identified the presence of specific targets; however, some Omicron variants, such as BA.4 and BA.5, couldn’t be distinguished from each other because they displayed the same pattern of results [ 32 ]. The same limitation in distinguishing Omicron variant groups is also seen in another recent study that is based on single-nucleotide polymorphism analysis, where only BA.1 and BA.2 sub-variants were included in the study [ 33 ].…”